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Protein Analytics Part I Protein Analytics Protein Purification Friedrich Lottspeich 1 Peter-Dörfler-Straße 4a, 82131 Stockdorf, Germany Investigation of the structure and function of proteins has already kept scientists busy for over 200 years. In 1777 the French chemist Pierre J. Macquer subsumed under the term Albumins all substances that showed the peculiar phenomenon of change from a liquid to a solid state upon warming. Chicken egg white, casein, and the blood component globulin already belonged to this class of substances. Already as early as 1787 (i.e., about the time of the French Revolution) the purification of egg white-like (coagulating) substances from plants was reported. In the early nineteenth century many proteins like albumin, fibrin, or casein were purified and analyzed. It soon became apparent that these compounds were considerably more complicated than other organic molecules known at that time. The word protein was most probably introduced by the Swedish chemist Jöns J. von Berzelius in about 1838 and was then published by the Dutch Gerardus J. Mulder. Mulder also suggested a chemical formula, which at that time was regarded as universally valid for all egg white-like materials. The homogeneity and purity of these purified proteins did not correspond of course to today’s demands. However, it became clear that proteins are different and distinct molecules. At that time purification could succeed only if one could use very simple steps, such as extraction for enrichment, acidification for precipitation, and spontaneous crystallization. Already in 1889 Hofmeister had obtained chicken albumin in crystalline form. Although Sumner in 1926 could already crystallize enzymatically active urease, the structure and the The molar mass (M) – often wrongly construction of proteins remained unknown up to the middle of the twentieth century. Only by called molecular weight – is not a mass the development of efficient purification methods, which allowed single proteins to be isolated but is defined as the mass of a substance from complicated mixtures, accompanied by a revolution in analysis techniques of the purified divided by the amount of substance: proteins, was today’s understanding of protein structures possible. M m=n NAmM This chapter describes fundamental purification methods and also touches on how they can be used systematically and strategically. It is extremely difficult to look at this subject in general The unit is g mol 1 terms, because the physical and chemical properties of single proteins may be very different. However, this structural diversity, which in the end determines also the function of the various Absolute molecule mass (mM) is the proteins, is biologically very meaningful and necessary. Proteins – the real tools and building molar mass of a molecule divided by the number of molecules in one mol materials of a cell – have to exercise a plethora of different functions. (= Avogadro constant, NA). mM = M/NA. The unit is g. The relative molecular mass (Mr)is 1.1 Properties of Proteins defined as the mass of one molecule normalized to the mass of 12C (carbon Size of Proteins The size of proteins can be very different. From small polypeptides, like 12), which by definition is equal to 12. insulin, which consists of 51 amino acids, up to very big multifunctional proteins, for example, to M 12 m =m 12 the apolipoprotein B, a cholesterol-transporting protein which contains more than 4600 amino r molecule C acid residues, with a molecular mass of more than 500 000 Dalton (500 kDa). Many proteins are It is dimensionless, but it has been composed of oligomers from the same or different protein chains and have molecule masses up to given the “unit” Dalton (Da) (formerly some millions Daltons. Quite in general it is to be expected that, the greater a protein is, the more atomic mass unit). Bioanalytics: Analytical Methods and Concepts in Biochemistry and Molecular Biology, First Edition. Edited by Friedrich Lottspeich and Joachim Engels. 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA. 4 Part I: Protein Analytics Figure 1.1 Separation methods of proteins and peptides. The separation capacity (i.e., the maximal number of compounds that can be separated in a single analysis) of the various separation methods is very different for different molecular masses of the analyte. Abbreviations: SEC, size exclusion chromatography; HIC, hydrophobic interaction chromatography; IEC, ion exchange chromatography; RPC, reversed phase chromatography; CE, capillary electrophoresis. Dalton (Da), named after the researcher difficultly its isolation and purification will be. This has its reason in the analytical procedures John Dalton (1766–1844), is a non-SI which show very low efficiencies with big molecules. Figure 1.1 shows the separation capacity mass unit. One Dalton is equivalent to (the maximum number of analytes, which can be separated under optimal condition) of individual = the atomic mass unit (u 1/12 of the separation techniques against the molecule mass of the analytes. 12 mass of C) and corresponds roughly It is evident that for small molecules like amino acids or peptides some chromatographic to the mass of one hydrogen atom (1.66 × 1024 g). In biochemistry the procedures are clearly able to distinguish more than 50 analytes in a single analysis. In the area > unit kDa (1 kilodalton = 1000 Da) is of proteins (Mr 104 Da) one recognizes that of the chromatographic techniques actually only very often used. ion exchange chromatography is able to separate efficiently more complicated mixtures. In the molecular mass area of proteins electrophoretic methods are by far more efficient. That is why Chromatographic Separation in proteome analysis (e.g., the analysis of all proteins of a cell), where several thousand proteins Techniques, Chapter 10 have to be separated, electrophoretic procedures (linear and two-dimensional) are very often used. From the figure is also evident that almost no efficient separation procedures exist for large molecules, for example, for protein complexes with molecular masses greater than Proteome Analysis, Chapter 39 150 kDa, or for organelles. The separation efficiency of a method is not always the relevant parameter in a protein purification. If selective purification steps are available the separation capacity is no longer significant and the selectivity becomes the crucial issue. Consequently, an affinity purification, which is based on the specific binding interaction of a substance to an affinity matrix, for example, an immune precipitation or an antibody affinity chromatography, has a quite low separation capacity of 1, but has an extremely high selectivity. Due to this highly selectivity a protein can easily be isolated even from a complex mixture in a one-step procedure. With the most common purification techniques, electrophoresis and chromatography, the analytes must be present in a dissolved form. Thus, the solubility of the protein in aqueous buffer media is a further important parameter when planning a protein purification. Many intracellular proteins, located in the cytosol (e.g., enzymes), are readily soluble while structure- forming proteins like cytoskeletal proteins or membrane proteins most often are much less soluble. Especially difficult to handle in aqueous solutions are hydrophobic integral membrane proteins, which are usually surrounded by lipid membranes. Without the presence of detergents Detergents, Section 1.8 such proteins will aggregate and precipitate during the purification. 1 Protein Purification 5 Available Quantity The quantity available in the raw material plays a crucial role in determining the effort that must be invested for a protein purification. A protein intended for the isolation is present perhaps only as a few copies per cell (e.g., transcription factors) or as a few thousand copies (e.g., many receptors). On the other hand, abundant proteins (e.g., enzymes) can constitute percentage shares of the total protein of a cell. Overexpressed proteins of proteins are often present in in clearly higher quantities (>50% in a cell) as well as some proteins in body fluids (e.g., albumin in plasma >60%). Purification with higher quantities of a protein is usually much simpler. Especially with the isolation of rare proteins different sources of raw material should be checked for the content of the protein of interest. Acid/Base Properties Proteins have certain acidic or basic properties because of their amino acid composition, properties that are used in separations via ion exchange chromatography and electrophoresis. The net charge of a protein is dependent on the pH of the surrounding solution. At a low pH-value it is positive, at high pH negative, and at the isoelectric point it is zero. Positive and negative charges compensate at the latter pH. Biological Activity The purification of a protein is often complicated by the fact that a particular protein often can be detected and localized among the various other proteins only due to its biological activity and location. Hence, one must take into account at every stage of protein isolation the preservation of this biological activity. Usually the biological activity is based on a specific molecular and spatial structure. If it is destroyed, one speaks of denaturation. This often is irreversible. To avoid denaturation, one must exclude in practice the application of Enzyme Activity Testing, some procedures. Chapter 3 The biological activity is often stable to different extents under different environmental conditions. Too high or too low buffer concentrations, temperature extremes, contacts with artificial surfaces such as glass or missing cofactors can change biological characteristics of proteins. Some of these changes are reversible: small proteins in particular are, after denatura- tion and loss of activity, often able to renature under certain conditions, regaining the biologically active form. For larger proteins, this is rarely the case and often results in only a poor yield. Measurement of the biological (e.g., enzymatic) activity makes it possible to monitor the purification of a protein.
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